In this project, we newly generated scores of novel non-peptidic PIs that exerted potent antiviral activity against both wild-type and drug-resistant HIV-1 variants. Included among such non-peptidic PIs are those containing a macrocycle and bis-THF. The inhibitors with macrocycles were designed to effectively fill in the hydrophobic pocket in the S10-S20 subsites and retain all major hydrogen bonding interactions with the protein backbone similar to DRV. The ring size, the effect of methyl substitution, and unsaturation within the macrocyclic ring structure were assessed. In general, cyclic inhibitors were significantly more potent than their acyclic homologues, saturated rings were less active than their unsaturated analogues and a preference for 10- and 13-membered macrocylic rings was revealed. The addition of methyl substituents resulted in a reduction of potency. Four macrocycle PIs exerted particularly potent activity against HIV-1LAI with an EC50 value of as low as 0.002 M with minimal cytotoxicity. Two such PIs, GRL-216 and -286, blocked the replication of HIV-1NL4-3 variants selected by up to 5 M of 5 existing FDA-approved PIs including lopinavir and atazanavir with EC50 values of 0.020 to 0.046 M and were potent against 6 multi-PI-resistant clinical HIV-1 variants (HIVmPIR) with EC50 values of 0.027-0.089 M. GRL-216 and -286 also blocked HIV-1 protease dimerization as efficiently as did DRV. HIV-1NL4-3 at passage 50 with GRL-216 (HIV216-0.16mM), containing L10I, L24I, M46L, V82I, and I84V mutations, remained relatively sensitive to PIs including DRV with EC50 values 3- to 8-fold-greater compared to each EC50 value to HIV1NL4-3. Analysis of the protein-ligand X-ray structures of GRL-216 revealed that the macrocycle occupied more volume of the binding cavity of protease and formed greater van der Waals interactions with V82 and I84 than DRV. The present data warrant that GRL-216 be further developed as a potential antiviral agent for treating individuals harboring wild-type and/or HIVmPIR. We previously identified a new class of nucleoside reverse transcriptase inhibitors (NRTIs), 4'-ethynyl-2'-deoxunucleoside analogs (EdNs), which, unlike conventional NRTIs such as azidothymidine, contain the 3'-hydroxyl moiety. These EdNs exert potent antiviral activity against a wide spectrum of HIV-1 and HIV-2 strains. In the time period of this annual report, we further examined the in vivo activity of 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA), one of the EdNs, which has a potent activity against various HIV-1 and simian immunodeficiency virus (SIV) strains including multi-drug resistant HIV-1 variants and is thought to be suitable for once daily regimen, in two SIV-infected monkeys, in collaboration with Dr. Michael Parniak of University of Pittsburgh. EFdA treatment resulted in a 2-3 log decrease in SIV load within 7 days. In further collaboration with Dr. Stefan Sarafianos of University of Missouri, the mechanism of the potent anti-HIV-1 activity was determined. It was found that the reverse transcriptase (RT) uses EFdA-5'-triphosphate (EFdA-TP) as substrate more efficiently than the natural substrate dATP. Importantly, despite the presence of a 3'-hydroxyl moiety, the incorporated EFdA-MP acts as a de facto terminator of further RT-catalyzed DNA synthesis, due to difficulty of RT translocation of the nucleic acid primer possessing 3'-terminal EFdA-MP. EFdA-TP is thus a Translocation Defective RT Inhibitor (TDRTI). This diminished translocation keeps the primer 3'-terminal EFdA-MP ideally located to undergo phosphorolytic excision. However, net phosphorolysis is not substantially increased due to apparently facile reincorporation of the newly excised EFdA-TP. Molecular modeling studies suggest that the 4'-ethynyl moiety fits into a hydrophobic pocket defined by RT residues A114, F160, M184 and D185. These interactions contribute both to enhanced RT utilization of EFdA-TP and difficulty in translocation of 3'-terminal EFdA-MP primers, and underlie the novel mechanism of action of this potent antiviral nucleoside. Virtual screening has been successful in the discovery of certain novel inhibitors, and a number of inhibitors have advanced to clinical trials. When the structure of a target protein is available, virtual screening involves docking potential inhibitors against the protein and ranking the inhibitors by their predicted affinity using a scoring function. Molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) or Molecular Mechanics Generalized Born surface area (MM-GBSA) have been used in some instances in the post-processing and re-ranking of results from molecular docking. Of note, docking and scoring have currently been an integral part of drug discovery efforts and have produced documented successes, however, there is an urgent need for improvement of the accuracy of docking and scoring results. With this regard, Clark has described four areas of improvement, i.e., better scoring functions, treatment of protein flexibility, treatment of water molecules, and improved technology for data analysis of virtual screening results. The scoring functions fail if they do not properly account for solvation, entropy, or polarizability. We have simulated the binding interactions and free energy of binding of nine protease inhibitors (PIs) with wild-type and various mutant proteases by performing GBSA simulations, in which each PIs partial charge was determined by quantum mechanics (QM) and the partial charge accounts for the polarization induced by the protease environment. We employed a hybrid solvation model that retains selected explicit water molecules in the protein with surface generalized Born (SGB) implicit solvent. We examined the correlation of the free energy with antiviral potency of PIs with regard to amino acid substitutions in protease. The GBSA free energy thus simulated showed strong correlations (r greater than 0.75) with antiviral IC50 values of PIs when amino acid substitutions were present in the protease active site. We also simulated the binding free energy of PIs with P2-bis-tetrahydrofuranylurethane (bis-THF) or related cores, utilizing a bis-THF-containing protease crystal structure as a template. The free energy showed a strong correlation (r = 0.93) with experimentally determined anti-HIV-1 potency. The present data suggest that the presence of selected explicit water in protein, and protein polarization-induced quantum charges for the inhibitor, compared to lack of explicit water and a static force field-based charge model, can serve as an improved lead optimization tool, and warrants further exploration.